Aedes aegpyti is a major vector of dengue, yellow fever, and chikungunya viruses. According to the World Health Organization, the global incidence of dengue has increased dramatically in recent decades and nearly half of the world population is now at risk. There is no specific treatment for dengue and current prevention depends solely on effective vector control. However, vector control methods are under threat as insecticide-resistance increases. New strategies to combat dengue and other mosquito-borne diseases are urgently needed. Several genetic strategies to suppress or replace vector populations are being explored. Since only female mosquitoes bite and transmit pathogens, the release of males is preferred and possibly required for implementation of these strategies. A better understanding of the sex-determination pathway will provide novel targets for interference and lead to several practical applications. For example, transgenic lines may be obtained that confer conditional female lethality, which will provide more cost-effective and scalable production of male-only mosquitoes than current approaches. Release of such transgenic males is more effective at achieving population reduction than sterile male release. However, little is known about sex determination in mosquitoes except for a few highly conserved genes such as doublesex (dsx) and transformer2 (tra2). Male development in Aedes mosquitos is initiated by a dominant male-determining locus (M-locus) located on the chromosome 1. However, no M-locus genes have been reported. We recently identified a novel male-specific gene in Ae. aegypti, which we named nix. Physical mapping places nix at the M-locus and nix genomic DNA was never detected in any female. No recombination was detected between nix and the M-locus when more than 5000 offspring were screened. Nix is expressed in the early embryo, larvae, pupae, and adult male testis, but not in any adult female tissues. Furthermore, the predicted NIX protein is an RNA-binding protein which may be involved in splicing. We hypothesize that nix plays an important role in sexual differentiation in Ae. aegypti. To test the hypothesis during thi exploratory R21 project, we will determine the effect of both ectopic expression (Aim 1) and knockout (Aim 2) of the nix gene. To determine the outcome of the two complementary approaches, we will monitor general phenotypic and molecular changes and pay close attention to those related to sexual differentiation. We anticipate extending our research beyond this R21 to investigate the mechanism of nix function and develop mosquito control applications through manipulation of the nix gene or its partners.